In vitro studies with hyperphosphorylated tau from either AD brain or generated in vitro show that this tau binds normal MAPs, disrupts microtubules, and self-assembles into PHFs/SFs (Alonso et al., 1994, 1996, 1997, 2001a, 2001b, 2004). These data provides compelling evidence to propose the following: a) in affected neurons, there is a phosphorylation imbalance;b) hyperphosphorylated tau binds MAPs both to disrupt microtubules and to self-assemble into filaments;c) this microtubule disruption interrupts axonal transport, preventing vesicular transport to the synapse;and d) this interruption results in the slow and steady degeneration of the synapse. Establishing the in vivo relevance of these in vitro results and of different tau phosphorylation sites will provide an explanation of the progression of tauopathies and it will identify new therapeutic targets for neurodegenerative diseases. It is proposed that the abnormal hyperphosphorylation of tau leads to neurofibrillary degeneration by cytoskeletal disruption, and that tau mutations associated with dementia promote their phosphorylation in vivo. The effect of tau hyperphosphorylation upon the cytoskeleton and neurodegeneration in cells in an in vivo-like system, will be investigated as follows: 1) What is the function of tau phosphorylation at positions 199, 212, 231, 235, 262, 396 individually or in combination on tau function, particularly for promoting microtubule activity, for modulating its ability to bind other MAPs, and for its ability to self-assembly? To approach an answer to this question tau phosphorylation at these positions will be mimicked by site directed mutagenesis changing Ser/Thr for Glu at these sites individually or in combination;the pseudophosphorylated forms of tau will be expressed in bacteria and purifyed and their ability to self-assembly, promote and inhibit the promotion of microtubule and its binding to normal tau, MAP1A, MAP1B and MAP2 will be studied. These studies will help understand the influence of tau phosphorylation at different phosphorylation sites. 2) What is the effect of expressing pseudophosphorylated tau in Drosophila's neurons? We plan to use Drosophila as a model system to study effects of peudohyperphosphorylated tau on neurofibrillary degeneration at multiple levels. Transgenic flies expressing pseudohyperphosphorylated tau with Thr212, Ser231, and Ser262 mutated individually or in combination by site-directed mutagenesis to Glu will be generated. The expression will be controlled temporally and spatially so that acute effects on specific type of neurons can be examined and to prevent developmental perturbations. The effect of tau hyperphosphorylation will be examined in axonal transport, neurodegeneration, and learning and memory. To establish parameters for modifier screening, eye degeneration and life-span will also be examined. Some of the proposed studies require little training and can be served as projects for high school and undergraduate students. PUBLIC HEALTH RELEVANCE: The information gained from these studies will provide insight into what different phosphorylation sites on tau are doing in the process of neurofibrillary degeneration, to explain the progression of tauopathies and identify new therapeutic targets for neurodegenerative diseases. The generation of pseudophosphorylated transgenic flies will provide a useful model of neurofibrillary degeneration for future studies, for drug screenings and for genetic modifiers screening.